The present invention is directed to enzymes, methods to purify, and obtain such enzymes, amino acid and nucleic acid sequences related thereto, and methods of use for such compositions in genetic engineering applications.
Through the development of plant genetic engineering techniques, it is possible to transform and regenerate a variety of plant species to provide plants which have novel and desirable characteristics. One area of interest for such plant genetic engineering techniques is the production of valuable products in plant tissues. Such applications require the use of various DNA constructs and nucleic acid sequences for use in transformation events to generate plants which produce the desired product. For example, plant functional promoters are required for appropriate expression of gene sequences, such expression being either in the whole plant or in selected plant tissues. In addition, selective marker sequences are often used to identify the transformed plant material. Such plant promoters and selectable markers provide valuable tools which are useful in obtaining the novel plants.
One desirable goal, which involves such genetic engineering techniques, is the ability to provide crop plants having a convenient source of wax esters. Wax esters are required in a variety of industrial applications, including pharmaceuticals, cosmetics, detergents, plastics, and lubricants. Such products, especially long chain wax esters, have previously been available from the sperm whale, an endangered species, or more recently, from the desert shrub, jojoba. Neither of these sources provides a convenient supply of wax esters.
Jojoba is also a plant which synthesizes very long chain fatty acids (VLCFA) in its seed oil. VLCFA are fatty acids having chain lengths longer than 18 carbons. VLCFA are found in the cuticular xe2x80x9cwaxesxe2x80x9d of many plant species as well as in the seed oil of several plant species. Wild type Brassica plants contain VLCFA in their seed oil. Canola is rapeseed that has been bred to eliminate VLCFA from its seed oil. Enzymes involved in the elongation of fatty acids to VLCFA (xe2x80x9celongasexe2x80x9d enzymes) have been difficult to characterize at a biochemical level because they are membrane associated (Harwood, J L, xe2x80x9cFatty acid metabolismxe2x80x9d, Annual rev. of Plant Physiol, and Plant Mal. Biol. (1988) 39:101-38); (von Wettstein-Knowles, P M, xe2x80x9cWaxes, cutin, and suberinxe2x80x9d in ed. Moore, T S, Lipid Metabolism in Plants (1993), CRC Press, Ann Arbor, pp. 127-166). Although several groups have claimed to partially purify some of these elongase enzymes, to date no one has claimed complete purification of one of these enzymes or cloning of the corresponding genes. von Wettstein-Knowles, P M, (1993) supra; van de Loo, F J, Fox, H G, and Somerville C. xe2x80x9cUnusual fatty acidsxe2x80x9d in ed. Moore, T S, Lipid Metabolism in Plants, (1993) CRC Press Ann Arbor, pp. 91-126.
A possible mechanism for fatty acid elongation by the cytoplasmic elongase enzyme system is through a series similar to that found for chloroplast fatty acid synthesis, i.e. via a 4 step reaction (Stumpf and Pollard (1983) supra; van de Loo et al (1993) supra). The first step would be a condensation reaction between malonyl CoA and oleyl CoA by xcex2-ketoacyl-CoA synthase. Then xcex2-ketoacyl-CoA reductase, xcex2-hydroxyacyl-CoA dehydratase, and enoyl-CoA reductase ensymes would act sequentially to generate an acyl-CoA molecule elongated by two carbon atoms.
In order to obtain a reliable source of very long chain fatty acid molecules, such as wax esters or VLCFA, transformation of crop plants, which are easily manipulated in terms of growth, harvest and extraction of products, is desirable. In order to obtain such transformed plants, however, the genes responsible for the biosynthesis of the desired VLCFA or wax ester products must first be obtained.
Wax ester production results from the action of at least two enzymatic activities of fatty acyl CoA metabolism; fatty acyl reductase and fatty acyl;fatty alcohol acyltransferase, or wax synthase. Preliminary studies with such enzymes and extensive analysis and purification of a fatty acyl reductase, indicate that these proteins are associated with membranes, however the enzyme responsible for the fatty acyl:fatty alcohol ligation reaction in wax biosynthesis has not been well characterized. Thus, further study and ultimately, purification of this enzyme is needed so that the gene sequences which encode the enzymatic activity may be obtained.
It is desirable, therefore, to devise a purification protocol whereby the wax synthase protein may be obtained and the amino acid sequence determined and/or antibodies specific for the wax synthase obtained. In this manner, library screening, polymerase chain reaction (PCR) or immunological techniques may be used to identify clones expressing a wax synthase protein. Clones obtained in this manner can be analyzed so that the nucleic acid sequences corresponding to wax synthase activity are identified. The wax synthase nucleic acid sequences may then be utilized in conjunction with fatty acyl reductase proteins, either native to the transgenic host cells or supplied by recombinant techniques, for production of wax esters in host cells.
It would also be desirable to have a gene to an enzyme involved in the formation of very long chain fatty acids. Such a gene could be used to increase the chain length of fatty acids in oilseeds by overexpression of the gene in transgenic plants of virtually any species. The gene could also be used as a probe in low stringency hybridization to isolate homologous clones from other species as a means to clone the gene from other taxa, such as Brassica, Arabidopsis, Crambe, Nasturtium, and Limnanthes, that produce VLCFA. These derived genes could then be used in antisense experiments to reduce the level of VLCFA in the species from which they were isolated, or overexpressed to increase the quantity of VLCFA in transgenic plants of virtually any species. Additionally, the DNA from the homologous Brassica gene encoding this enzyme could be used as a plant breeding tool to develop molecular markers to aid in breeding high erucic acid rapeseed (HEAR) and canola and other oilseed crops. Such techniques would include using the gene itself as a molecular probe or using the DNA sequence to design PCR primers to use PCR based screening techniques in plant breeding programs. Finally, overexpression of the gene in plant epidermal cells could increase cuticle accumulation thereby increasing drought and stress tolerance of transgenic plants over control plants.
Relevant Literature
Cell-free homogenates from developing jojoba embryos were reported to have acyl-CoA fatty alcohol acyl transferase activity. The activity was associated with a floating wax pad which formed upon differential centrifugation (Pollard et al. (1979) supra; Wu et al. (1981) supra).
Solubilization of a multienzyme complex from Euglena gracilis having fatty acyl-SCoA transacylase activity is reported by Wildner and Hallick (Abstract from The Southwest Consortium Fifth Annual Meeting, April 22-24, 1990, Las Cruces, N.Mex.).
Ten-fold purification of jojoba acyl-CoA: alcohol transacylase protein is reported by Pushnik et al. (Abstract from The Southwest Consortium Fourth Annual Meeting, Feb. 7, 1989, Riverside, Calif.).
An assay for jojoba acyl-CoA: alcohol transacylase activity was reported by Garver et al. (Analytical Biochemistry (1992) 207:335-340).
Extracts of developing seeds from HEAR and canola plants were found to differ in their ability to elongate oleyl CoA into VLCFA, with HEAR extracts capable of catalyzing elongation, while canola extracts were not. Stumpf, P K and Pollard M R, xe2x80x9cPathways of fatty acid biosynthesis in higher plants with particular reference to developing rapeseedxe2x80x9d, in High and Low Erucic Acid Rapeseed Oils (1983) Academic Press Canada, pp. 131-141.